Characterization of Diversity and Probiotic Efficiency of the Autochthonous Lactic Acid Bacteria in the Fermentation of Selected Raw Fruit and Vegetable Juices

Abstract

The diversity of indigenous lactic acid bacteria (LAB) in fermented broccoli, cherry, ginger, white radish, and white-fleshed pitaya juices was analyzed using culture-independent and -dependent approaches. The major properties of selected probiotic strains, including dynamic variations in pH, viable cell counts, antibiotic resistance, bacterial adhesion to hydrophobic compounds, and survivability during simulated gastrointestinal transit, were investigated using broccoli as the fermentation substrate. In broccoli and ginger juices, the genus Lactobacillus occupied the dominant position (abundances of 79.0 and 30.3%, respectively); in cherry and radish juices, Weissella occupied the dominant position (abundances of 78.3 and 83.2%, respectively); and in pitaya juice, Streptococcus and Lactococcus occupied the dominant positions (52.2 and 37.0%, respectively). Leuconostoc mesenteroides, Weissella cibaria/soli/confusa, Enterococcus gallinarum/durans/hirae, Pediococcus pentosaceus, Bacillus coagulans, and Lactococcus garvieae/lactis subspecies were identified by partial 16S rRNA gene sequencing. In general, the selected autochthonous LAB isolates displayed no significant differences in comparison with commercial strains with regard to growth rates or acidification in fermented broccoli juice. Among all the isolates, L. mesenteroides B4-25 exhibited the highest antibiotic resistance profile (equal to that of L. plantarum CICC20265), and suitable adhesion properties (adhesion of 13.4 ± 5.2% ∼ 36.4 ± 3.2% and 21.6 ± 1.4% ∼ 69.6 ± 2.3% to ethyl acetate and xylene, respectively). Furthermore, P. pentosaceus Ca-4 and L. mesenteroides B-25 featured the highest survival rates (22.4 ± 2.6 and 21.2 ± 1.4%, respectively), after simulated gastrointestinal transit. These results indicated a high level of diversity among the autochthonous bacterial community in fermented fruit and vegetable juices, and demonstrated the potential of these candidate probiotics for applications in fermentation.

Keywords: autochthonous lactic acid bacteria; fermentation; fruit and vegetable juice; microbial diversity; probiotic viability.

FIGURE 1

Distribution of the microbial community for each sample at the genus level. The data were visualized by Circos. The small semi-circle (left) shows the composition and abundance of species in each sample. The large semi-circle (right) shows the distribution ratio of samples within the dominant species, and the width of the bars for each genus indicates the relative abundance of that genus in the sample. From the outer circle to the inner circle, the outer circle shows the different samples and species using specific colors, and the length is related to the distribution proportion. The second circle shows the percentage. In the innermost circle, the two ends of each chromatic stripe connect the sample with the dominant genus; the width of the stripe at the endpoint represents the abundance and distribution proportion, and the numerical value outside the circle represents the abundance values of the corresponding samples and species.

FIGURE 2

Variance analysis of the bacterial communities among the fermented juices. (A) PCA plots indicate the abundance of diverse bacteria in the fermented juices. The first principal component (PC1) and second principal component (PC2) shows 26.65 and 60.09%, respectively, of the variance in the unweighted Unifrac metrics. Each point represents the microbiota from a single sample. (B) Hierarchical clustering of the group means based on UPGMA.

FIGURE 3

Comparison of dominant genera in five samples using one-way ANOVA. In the vertical axis, the different identified bacteria genera are depicted; the length of the corresponding column indicates the average of the relative abundance of the genus in the different samples, ^∗∗P < 0.01, ^∗P < 0.05; NA, not available.

FIGURE 4

Phylogenetic tree constructed with sequences of the partial 16S rRNA gene of selected LAB strains. The 16S sequences of the isolated bacteria are aligned with reference strains. The data of type strains of related species were from GenBank database. Branch lengths are proportional to distance. Bootstrap values are indicated near the internodes. B, R, C, G, and P represent juices of broccoli, radish, cherry, ginger, and pitaya, respectively; Ba, Ra, Ca, Ga, and Pa represent juices of broccoli, radish, cherry, ginger, and pitaya after fermentation, respectively.

FIGURE 5

Viable cell counts and pH values throughout the fermentation of broccoli juices incubated with the nine selected LAB strains. (A,B) Represent the change in viable cell counts and pH, respectively.

FIGURE 6

Evaluation of hydrophobicity to ethyl acetate and xylene and survival of simulated gastrointestinal digestion of the nine selected LAB strains. (A) Percentage of hydrophobicity. (B) Survival capacity of LAB strains during simulated gastrointestinal digestion. GJ, gastric juice; DJ, duodenal juice; IJ, intestinal juice.